Formation and function of a highly specialised type of organelle in cardiac valve cells.

Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. In this study, we have analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. We show that the compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells.

Alary muscles and thoracic alary-related muscles are atypical striated muscles involved in maintaining the position of internal organs.

Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side and internal organs on the other, suggested an architectural function in moving larvae. Here, we analysed the shape and sarcomeric organisation of AMs and TARMs, and imaged their atypical deformability in crawling larvae. We then selectively eliminated AMs and TARMs by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-haemocelic position and for opening of the heart lumen, and that AMs constrain the curvature of the respiratory tracheal system during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions.

Does a brief surgeon training in negotiation theory principles decrease rates of contralateral prophylactic mastectomy?

PURPOSE: Despite the lack of any oncologic benefit, contralateral prophylactic mastectomy (CPM) use among women with unilateral breast cancer is increasing. This patient-driven trend is influenced by fear of recurrence and desire for peace of mind. Traditional educational strategies have been ineffective in reducing CPM rates. Here we employ training in negotiation theory strategies for counseling and determine the effect on CPM rates. METHODS: In consecutive patients with unilateral breast cancer treated with mastectomy from 05/2017 to 12/2019, we examined CPM rates before and after a brief surgeon training in negotiation skills. This comprised a systematic framework for patient counseling utilizing early setting of the default option, leveraging social proof, and framing. RESULTS: Among 2144 patients, 925 (43%) were treated pre-training and 744 (35%) post-training. Those treated in the 6-month transition period were excluded (n = 475, 22%). Median patient age was 50 years; most patients had T1-T2 (72%), N0 (73%), and estrogen receptor-positive (80%) tumors of ductal histology (72%). The CPM rate was 47% pre-training versus 48% post-training, with an adjusted difference of -3.7% (95% CI -9.4 to 2.1, p = 0.2). In a standardized self-assessment survey, all 15 surgeons reported a high baseline use of negotiation skills and no significant change in conversational difficulty with the structured approach. CONCLUSION: Brief surgeon training did not affect self-reported use of negotiation skills or reduce CPM rates. The choice of CPM is a highly individual decision influenced by patient values and decision styles. Further research to identify effective strategies to minimize surgical overtreatment with CPM is needed.

Local Recurrence is Frequent After Heroic Mastectomy for Classically Inoperable Breast Cancers.

BACKGROUND: Despite advances in neoadjuvant systemic therapy (NST), some patients with aggressive T4 breast cancers do not respond. The efficacy of 'heroic' mastectomy in maintaining local control is unclear. METHODS: In consecutive patients with primary or recurrent T4 cancers with < 50% shrinkage on NST who underwent mastectomy from 2007 to 2017, clinicopathologic characteristics and locoregional recurrence (LRR) were examined. RESULTS: Among 104 patients, 59 (57%) had primary T4M0, 12 (12%) had locally recurrent T4M0, and 33 (32%) had T4M1 disease. Median age was 58.5 years and the majority had high-grade (74%) ductal cancers (85%); 45 (44%) were estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+/HER2-), 26 (25%) were HER2 positive (HER2+), and 31 (30%) were triple negative (TN). Postoperative complications developed in 41 (39%) patients. At a median follow-up of 37 months, 42 (40%) patients developed LRR. TN (hazard ratio [HR] 7.5) and HER2+ (HR 2.67) subtypes, lymphovascular invasion (LVI; HR 3.80), and positive margins (HR 4.09) were predictive of LRR. The 3-year LRR rate was highest and overall survival (OS) was lowest among patients with TN cancers, at 66% (95% confidence interval [CI] 48-83%) and 30% (95% CI 14-47%), respectively. CONCLUSIONS: After heroic mastectomy, postoperative complications were frequent and LRR occurred in 40% of patients despite a median OS of 3.8 years. Among TN patients, the 3-year LRR rate of 66% and 3-year OS of 30% suggest limited surgery benefit. Careful patient selection is prudent when considering heroic mastectomy.

Adoption of SSO-ASTRO Margin Guidelines for Ductal Carcinoma in Situ: What Is the Impact on Use of Additional Surgery?

BACKGROUND: Historically, more than one-third of patients with ductal carcinoma in situ (DCIS) treated with breast-conserving surgery (BCS) underwent additional surgery. The SSO-ASTRO guidelines advise 2 mm margins for patients with DCIS having BCS and whole-breast radiation (WBRT). Here we examine guideline impact on additional surgery and factors associated with re-excision. PATIENTS AND METHODS: Patients treated with BCS for pure DCIS from August 2015 to January 2018 were identified. Guidelines were adopted on September 1, 2016, and all patients had separately submitted cavity-shave margins. Clinicopathologic characteristics, margin status, and rates of additional surgery were examined. RESULTS: Among 650 patients with DCIS who attempted BCS, 50 (8%) converted to mastectomy. Of 600 who had BCS as final surgery, 336 (56%) received WBRT and comprised our study group. One hundred twenty-eight (38%) were treated pre-guideline and 208 (62%) were treated post-guideline. Characteristics and margin status were similar between groups. The re-excision rate was 38% pre-guideline adoption and 29% post-guideline adoption (p = 0.09), with 91% having only one re-excision. Re-excision for ≥ 2 mm margins was uncommon (6% pre-guideline vs. 5% post-guideline). On multivariate analysis, younger age (OR 0.97, 95% CI 0.94-0.99, p = 0.02) and larger DCIS size (OR 1.43, 95% CI 1.2-1.8, p < 0.001) were predictive of re-excision; guideline era was not. Younger age (OR 0.93, 95% CI 0.9-0.97, p < 0.001) and larger size (OR 1.64, 95% CI 1.3-2.1, p < 0.001) were predictive of conversion to mastectomy, but residual tumor burden was low. CONCLUSIONS: The SSO-ASTRO guidelines did not significantly change re-excision rates for DCIS in our practice, likely since re-excision for margins ≥ 2 mm was uncommon even prior to guideline adoption, dissimilar to historically observed variations in surgeon practices. Younger age and larger DCIS size were associated with additional surgery.

Extranodal Tumor Deposits in the Axillary Fat Indicate the Need for Axillary Dissection Among T1-T2cN0 Patients with Positive Sentinel Nodes.

BACKGROUND: The American College of Surgeons Oncology Group (ACOSOG) Z0011 trial demonstrated the safety of omitting axillary lymph node dissection (ALND) in T1-T2cN0 patients with fewer than three positive sentinel nodes (SLNs) undergoing breast-conservation therapy. While microscopic extracapsular extension (mECE) > 2 mm is associated with increased nodal burden, the significance of extranodal tumor deposits (ETDs) in the axillary fat is uncertain. METHODS: Consecutive patients with T1-T2cN0 breast cancer undergoing sentinel node biopsy and ALND for SLN metastases from January 2010 to December 2018 were identified. ETDs were defined as intravascular tumor emboli or metastatic deposits in the axillary fat. Clinicopathologic characteristics and nodal burden were compared by ETD status. RESULTS: Among 1114 patients, 113 (10%) had ETDs: 81 (72%) were intravascular tumor emboli and 32 (28%) were soft tissue deposits. Patients with ETDs had larger tumors (median 2.2 vs. 2.1 cm; p = 0.033) and more often had mECE (83% vs. 44%; p < 0.001). On univariable analysis, presence of ETDs (odds ratio [OR] 9.66, 95% confidence interval [CI] 6.36-14.68), larger tumors (OR 1.47, 95% CI 1.25-1.72), and mECE (OR 10.73, 95% CI 6.86-16.78) were associated with four or more additional positive non-SLNs (NSLNs; all p < 0.001). On multivariable analysis, ETDs remained associated with four or more positive NSLNs (OR 5.67, 95% CI 3.53-9.08; p < 0.001). ETDs were strongly associated with four or more positive NSLNs (OR 7.15, 95% CI 4.04-12.67) among patients with one to two positive SLNs (n = 925). CONCLUSIONS: Among T1-T2cN0 patients with SLN metastases, ETDs are strongly associated with four or more positive NSLNs at ALND. Even among those who may otherwise meet the criteria for omission of ALND, the presence of ETDs in axillary fat warrants consideration of ALND.

Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles.

BACKGROUND: A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs) expressed by its FC. Here, we addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? RESULTS: The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. CONCLUSIONS: Transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.

An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila.

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates.

Tup/Islet1 integrates time and position to specify muscle identity in Drosophila.

The LIM-homeodomain transcription factor Tailup/Islet1 (Tup) is a key component of cardiogenesis in Drosophila and vertebrates. We report here an additional major role for Drosophila Tup in specifying dorsal muscles. Tup is expressed in the four dorsal muscle progenitors (PCs) and tup-null embryos display a severely disorganized dorsal musculature, including a transformation of the dorsal DA2 into dorsolateral DA3 muscle. This transformation is reciprocal to the DA3 to DA2 transformation observed in collier (col) mutants. The DA2 PC, which gives rise to the DA2 muscle and to an adult muscle precursor, is selected from a cluster of myoblasts transiently expressing both Tinman (Tin) and Col. The activation of tup by Tin in the DA2 PC is required to repress col transcription and establish DA2 identity. The transient, partial overlap between Tin and Col expression provides a window of opportunity to distinguish between DA2 and DA3 muscle identities. The function of Tup in the DA2 PC illustrates how single cell precision can be reached in cell specification when temporal dynamics are combined with positional information. The contributions of Tin, Tup and Col to patterning Drosophila dorsal muscles bring novel parallels with chordate pharyngeal muscle development.

Size control of the Drosophila hematopoietic niche by bone morphogenetic protein signaling reveals parallels with mammals.

The Drosophila melanogaster larval hematopoietic organ, the lymph gland, is a model to study in vivo the function of the hematopoietic niche. A small cluster of cells in the lymph gland, the posterior signaling center (PSC), maintains the balance between hematopoietic progenitors (prohemocytes) and their differentiation into specialized blood cells (hemocytes). Here, we show that Decapentaplegic/bone morphogenetic protein (Dpp/BMP) signaling activity in PSC cells controls niche size. In the absence of BMP signaling, the number of PSC cells increases. Correlatively, no hemocytes differentiate. Controlling PSC size is, thus, essential for normal blood cell homeostasis. Activation of BMP signaling in the PSC requires expression of the Dally-like heparan-sulfate proteoglycan, under the control of the Collier/early B-cell factor (EBF) transcription factor. A Dpp > dpp autoregulatory loop maintains BMP signaling, which limits PSC cell proliferation by repressing the protooncogene dmyc. Dpp antagonizes activity of wingless (Wg)/Wnt signaling, which positively regulates the number of PSC cells via the control of Dmyc expression. Together, our data show that Collier controls hemocyte homeostasis via coordinate regulation of PSC cell number and PSC signaling to prohemocytes. In mouse, EBF2, BMP, and Wnt signaling in osteoblasts is required for the proper number of niche and hematopoietic stem cells. Our findings bring insights to niche size control and draw parallels between Drosophila and mammalian hematopoiesis.

A Real-World Assessment of Stage I Lung Cancer Through Electronic Nose Technology.

INTRODUCTION: Electronic nose (E-nose) technology has reported excellent sensitivity and specificity in the setting of lung cancer screening. However, the performance of E-nose specifically for early-stage tumors remains unclear. Therefore, the aim of our study was to assess the diagnostic performance of E-nose technology in clinical stage I lung cancer. METHODS: This phase IIc trial (NCT04734145) included patients diagnosed with a single greater than or equal to 50% solid stage I nodule. Exhalates were prospectively collected from January 2020 to August 2023. Blinded bioengineers analyzed the exhalates, using E-nose technology to determine the probability of malignancy. Patients were stratified into three risk groups (low-risk, [<0.2]; moderate-risk, [≥0.2-0.7]; high-risk, [≥0.7]). The primary outcome was the diagnostic performance of E-nose versus histopathology (accuracy and F1 score). The secondary outcome was the clinical performance of the E-nose versus clinicoradiological prediction models. RESULTS: Based on the predefined cutoff (<0.20), E-nose agreed with histopathologic results in 86% of cases, achieving an F1 score of 92.5%, based on 86 true positives, two false negatives, and 12 false positives (n = 100). E-nose would refer fewer patients with malignant nodules to observation (low-risk: 2 versus 9 and 11, respectively; p = 0.028 and p = 0.011) than would the Swensen and Brock models and more patients with malignant nodules to treatment without biopsy (high-risk: 27 versus 19 and 6, respectively; p = 0.057 and p < 0.001). CONCLUSIONS: In the setting of clinical stage I lung cancer, E-nose agrees well with histopathology. Accordingly, E-nose technology can be used in addition to imaging or as part of a "multiomics" platform.

Combinatorial coding of Drosophila muscle shape by Collier and Nautilus.

The diversity of Drosophila muscles correlates with the expression of combinations of identity transcription factors (iTFs) in muscle progenitors. Here, we address the question of when and how a combinatorial code is translated into muscle specific properties, by studying the roles of the Collier and Nautilus iTFs that are expressed in partly overlapping subsets of muscle progenitors. We show that the three dorso-lateral (DL) progenitors which express Nautilus and Collier are specified in a fixed temporal sequence and that each expresses additionally other, distinct iTFs. Removal of Collier leads to changes in expression of some of these iTFs and mis-orientation of several DL muscles, including the dorsal acute DA3 muscle which adopts a DA2 morphology. Detailed analysis of this transformation revealed the existence of two steps in the attachment of elongating muscles to specific tendon cells: transient attachment to alternate tendon cells, followed by a resolution step selecting the final sites. The multiple cases of triangular-shaped muscles observed in col mutant embryos indicate that transient binding of elongating muscle to exploratory sites could be a general feature of the developing musculature. In nau mutants, the DA3 muscle randomly adopts the attachment sites of the DA3 or DO5 muscles that derive from the same progenitor, resulting in a DA3, DO5-like or bifid DA3-DO5 orientation. In addition, nau mutant embryos display thinner muscle fibres. Together, our data show that the sequence of expression and combinatorial activities of Col and Nau control the pattern and morphology of DL muscles.

Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo.

Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of 'identity' transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, we show here that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. We conclude that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity.

A short receptor downregulates JAK/STAT signalling to control the Drosophila cellular immune response.

The posterior signalling centre (PSC), a small group of specialised cells, controls hemocyte (blood cell) homeostasis in the Drosophila larval hematopoietic organ, the lymph gland. This role of the PSC is very reminiscent of the "niche," the micro-environment of hematopoietic stem cells in vertebrates. We have recently shown that the PSC acts in a non-cell-autonomous manner to maintain janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) signalling in hematopoietic progenitors (prohemocytes), thereby preserving the multipotent character necessary for their differentiation into lamellocytes, a cryptic and dedicated immune cell type required to fight specific immune threats such as wasp parasitism. In this report, on the basis of a knock out generated by homologous recombination, we show that a short type I cytokine-related receptor CG14225/Latran is required for switching off JAK/STAT signalling in prohemocytes. This is a prerequisite to massive differentiation of lamellocytes upon wasp parasitisation. In vivo and cell culture assays indicate that Latran forms heteromers with Domeless, the Drosophila type I cytokine signalling receptor related to mammalian GP130, and antagonises Domeless activity in a dose-dependent manner. Our analysis further shows that a primary immune response to wasp parasitism is a strong decrease in cytokine mRNA levels in the lymph gland, followed by an increase in the latran/domeless ratio. We propose that this sequence of events culminates in the complete inhibition of residual JAK/STAT signalling by Latran. JAK/STAT activity has been associated with several human diseases including leukaemia while knock-out studies in mice point to a central role of this pathway in hematopoiesis and regulation of immune functions. The specific function of Drosophila Latran is, to our knowledge, the first in vivo example of a role for a nonsignalling receptor in controlling a dedicated immune response, and thus raises the question of whether short, nonsignalling receptors also control specific aspects of vertebrate cellular immunity.

Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre.

Drosophila haemocytes (blood cells) originate from a specialized haematopoietic organ-the lymph gland. Larval haematopoietic progenitors (prohaemocytes) give rise to three types of circulating haemocytes: plasmatocytes, crystal cells and lamellocytes. Lamellocytes, which are devoted to encapsulation of large foreign bodies, only differentiate in response to specific immune threats, such as parasitization by wasps. Here we show that a small cluster of signalling cells, termed the PSC (posterior signalling centre), controls the balance between multipotent prohaemocytes and differentiating haemocytes, and is necessary for the massive differentiation of lamellocytes that follows parasitization. Communication between the PSC and haematopoietic progenitors strictly depends on the PSC-restricted expression of Collier, the Drosophila orthologue of mammalian early B-cell factor. PSC cells act, in a non-cell-autonomous manner, to maintain JAK/STAT signalling activity in prohaemocytes, preventing their premature differentiation. Serrate-mediated Notch signalling from the PSC is required to maintain normal levels of col transcription. The key role of the PSC in controlling blood cell homeostasis is reminiscent of interactions between haematopoietic progenitors and their micro-environment in vertebrates, thus further highlighting the interest of Drosophila as a model system for studying the evolution of haematopoiesis and cellular innate immunity.

Insights and perspectives on the enigmatic alary muscles of arthropods.

Three types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles.

Hematopoietic progenitors and hemocyte lineages in the Drosophila lymph gland.

The Drosophila lymph gland (LG) is a model system for studying hematopoiesis and blood cell homeostasis. Here, we investigated the patterns of division and differentiation of pro-hemocytes in normal developmental conditions and response to wasp parasitism, by combining lineage analyses and molecular markers for each of the three hemocyte types. Our results show that the embryonic LG contains primordial hematopoietic cells which actively divide to give rise to a pool of pro-hemocytes. We found no evidence for the existence of bona fide stem cells and rather suggest that Drosophila pro-hemocytes are regulated as a group of cells, rather than individual stem cells. The fate-restriction of plasmatocyte and crystal cell progenitors occurs between the end of embryogenesis and the end of the first larval instar, while Notch activity is required for the differentiation of crystal cells in third instar larvae only. Upon parasitism, lamellocyte differentiation prevents crystal cell differentiation and lowers plasmatocyte production. We also found that a new population of intermediate progenitors appears at the onset of hemocyte differentiation and accounts for the increasing number of differentiated hemocytes in the third larval instar. These findings provide a new framework to identify parameters of developmental plasticity of the Drosophila lymph gland and hemocyte homeostasis in physiological conditions and in response to immunological cues.

A Phase I Trial of Regional Mesothelin-Targeted CAR T-cell Therapy in Patients with Malignant Pleural Disease, in Combination with the Anti-PD-1 Agent Pembrolizumab.

Malignant pleural diseases, comprising metastatic lung and breast cancers and malignant pleural mesothelioma (MPM), are aggressive solid tumors with poor therapeutic response. We developed and conducted a first-in-human, phase I study of regionally delivered, autologous, mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Intrapleural administration of 0.3M to 60M CAR T cells/kg in 27 patients (25 with MPM) was safe and well tolerated. CAR T cells were detected in peripheral blood for >100 days in 39% of patients. Following our demonstration that PD-1 blockade enhances CAR T-cell function in mice, 18 patients with MPM also received pembrolizumab safely. Among those patients, median overall survival from CAR T-cell infusion was 23.9 months (1-year overall survival, 83%). Stable disease was sustained for ≥6 months in 8 patients; 2 exhibited complete metabolic response on PET scan. Combination immunotherapy with CAR T cells and PD-1 blockade agents should be further evaluated in patients with solid tumors. SIGNIFICANCE: Regional delivery of mesothelin-targeted CAR T-cell therapy followed by pembrolizumab administration is feasible, safe, and demonstrates evidence of antitumor efficacy in patients with malignant pleural diseases. Our data support the investigation of combination immunotherapy with CAR T cells and PD-1 blockade agents in solid tumors.See related commentary by Aldea et al., p. 2674.This article is highlighted in the In This Issue feature, p. 2659.

Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog.

We here identify and characterize an extracellular modulator of Hedgehog signaling in Drosophila, Shifted. Shifted is required for high levels of long-range signaling in the developing wing imaginal disc. Surprisingly, shifted encodes the only Drosophila ortholog of the secreted vertebrate protein Wnt Inhibitory Factor-1 (WIF-1), whose known role is to bind to extracellular Wnts and inhibit their activity. However, Shifted does not regulate Hedgehog signaling by affecting Wingless or Wnt signaling. We show instead that Shifted is a secreted protein that acts over a long distance and is required for the normal accumulation of Hh protein and its movement in the wing. Our data further indicate that Shf interacts with Hh and the heparan sulfate proteoglycans. Therefore, we propose that Shf stabilizes the interaction between Hh and the proteoglycans, an unexpected role for a member of the WIF-1 family.

Drosophila ammonium transporter Rh50 is required for integrity of larval muscles and neuromuscular system.

Rhesus glycoproteins (Rh50) have been shown to be ammonia transporters in many species from bacteria to human. They are involved in various physiological processes including acid excretion and pH regulation. Rh50 proteins can also provide a structural link between the cytoskeleton and the plasma membranes that maintain cellular integrity. Although ammonia plays essential roles in the nervous system, in particular at glutamatergic synapses, a potential role for Rh50 proteins at synapses has not yet been investigated. To better understand the function of these proteins in vivo, we studied the unique Rh50 gene of Drosophila melanogaster, which encodes two isoforms, Rh50A and Rh50BC. We found that Drosophila Rh50A is expressed in larval muscles and enriched in the postsynaptic regions of the glutamatergic neuromuscular junctions. Rh50 inactivation by RNA interference selectively in muscle cells caused muscular atrophy in larval stages and pupal lethality. Interestingly, Rh50-deficiency in muscles specifically increased glutamate receptor subunit IIA (GluRIIA) level and the frequency of spontaneous excitatory postsynaptic potentials. Our work therefore highlights a new role for Rh50 proteins in the maintenance of Drosophila muscle architecture and synaptic physiology, which could be conserved in other species.